This new project involves the 3D reconstruction of meiotic spindles, both meiosis I and meiosis II, from the budding yeast S. cerevisiae. The project is funded by a grant from the March of Dimes Birth Defects Foundation (FY98-0409). We have already completed a study of mitotic spindles in this organism and wish to compare their organization to that of meiotic spindles. In this yeast, the meiotic spindles are shorter than mitotic spindles, and we are interested to determine if the spindles are organized differently to accommodate this difference. We are also interested to know the nature of MI and MII anaphase A movements. Finally, the MI spindle is responsible for separating the homologs, whereas the MII spindles are responsible for separating the sister chromatids and we wish to know if this functional difference is reflected in any microtubule organizational differences between the two spindle types. We have used the high-pressure freezing and freeze substitution techniques developed for mitotic cells on cells in meiosis with outstanding success. The image quality is excellent and the detection of microtubules in cross-section as needed for modeling the spindles is quite easy. Thus far, we have built two models of meiotic spindles by manual microtubule tracking, and we do not anticipate any difficulties. In fact, the images are of the quality that automated microtubule tracking has been used successfully on one data set from a meiotic cell. That data set was generated using the new digital Gatan camera on the Philips CM10 microscope, and the image quality appears to be sufficient for microtubule tracking. The ability to go "direct to digital" with the Gatan camera and to use automated microtubule tracking should greatly accelerate our production of meiotic spindle models. D1